Robert F. Whitcomb

Plant Mycoplasmas: A Cultivable Spiroplasma Causes Corn Stunt Disease

A spiroplasma can be isolated and grown continuously in cell-free medium from stunted corn or from Drosophila injected with sap expressed from diseased corn. The organism is serologically related to, but not identical with, Spiroplasma citri, the causative agent of citrus stubborn disease. Leafhopers injected with cultured organisms induced typical symptoms of the corn stunt disease when placed on previously healthy corn plants.

The Spiroplasma Deformation Test, a New Serological Method

The deformation test is a simple and highly sensitive technique capable of demonstrating significant antigenic differences among helical, wall-less prokaryotes (spiroplasmas). Specific identified. Quantitative relationships among various antisera are determined by examining, under dark-field microscopy, samples containing serum dilutions and a measured number of organisms which is held constant in each test. Antisera dilutions of 1:2,000 to 1;16,000 deformed spiroplasmas in homologous tests involvingSpiroplasma citri and the corn stunt and suckling mouse cataract spiroplasmas. With the exception of some heterologous cross-reactions in the deformation test betweenS. citri and corn stunt spiroplasmas, antisera and preimmunization sera failed to deform heterologous spiroplasmas at dilutions higher than 1:16.

Serological relationships of spiroplasmas as shown by combined deformation and metabolism inhibition tests

The deformation test and the metabolism inhibition test are simple and highly sensitive serological tests capable of revealing antigenic differences among spiroplasmas. Spiroplasma strains from plant and invertebrate hosts were compared in a combined deformation-metabolism inhibition test conducted in microtiter plates. Four major serological groups of spiroplasmas were recognized on the basis of deformation-metabolism inhibition tests. Group 1 was the Spiroplasma citri complex, comprised of isolates from plants, insects, and ticks. This group was provisionally divided into four serological subgroups. Subgroup 1 included the S. citri type strain (Maroc) and spiroplasmas isolated by Kondo and associates from cactus and lettuce. The second subgroup contained three corn stunt spiroplasma isolates. Subgroup 3 comprised 2 Spiroplasma strains isolated from honey bees by T. B. Clark. Subgroup 4 consisted of a single isolate (277F) from ticks. Each of these subgroups showed some serological cross-reactions with one or more of the other subgroups. Three other serologically distinct Spiroplasma clusters were observed. The suckling mouse cataract agent and an additional rabbit tick isolate (TP-2) were placed in group 2. Group 3 spiroplasmas included T. B. Clarks isolates (OBMG and BNR1) from flowers of magnolia and tulip trees. The fourth distinct serological group was represented by the uncultivated sex ratio spiroplasmas from Drosophila.

Spiroplasma melliferum, a New Species from the Honeybee (Apis mellifera)

Twenty-eight strains of spiroplasma subgroup I-2 isolated from insects and flower surfaces were similar in their serological properties. Strain BC-3T (T = type strain), which was isolated from the honeybee, was chosen as a representative of this cluster and was characterized according to accepted standards. This strain and other strains of the cluster entered the hemocoel of their insect hosts after per os acquisition, caused pathology in various tissues, and reduced adult longevity. Growth in SM-1 or M1D medium occurred at 20 to 37°, with optimum growth at about 32 to 35°. Cholesterol was required for growth. Glucose, fructose, and other carbohydrates were fermented, and arginine was catabolized. Seventeen strains, including strain BC-3T, reacted with considerable homogeneity in deformation tests and were completely separable from strains of subgroup I-1 (Spiroplasma citri) and subgroup I-3 (corn stunt spiroplasma). A group of five subgroup I-2 strains showed homogeneity upon one-dimensional polyacrylamide gel electrophoresis of cell proteins. Strain BC-3T was also serologically distinct from subgroups I-4 through I-8; from Spiroplasma floricola, Spiroplasma apis, and Spiroplasma mirum; and from representative strains of spiroplasma groups II and VI through XI. Previously published studies on strain BC-3T and related strains demonstrated that (i) these organisms comprise a unique subgroup of the S. citri complex (group I); (ii) deoxyribonucleic acid-deoxyribonucleic acid homologies between strain BC-3T and strains of other group I subgroups do not exceed 70%; (iii) the patterns of protein sharing among group I strains revealed by two-dimensional polyacrylamide gel electrophoresis support molecular genetic indications of partial relatedness; (iv) the EcoRI restriction endonuclease patterns of deoxyribonucleic acids from strain BC-3T and serologically related strains show close relatedness; (v) sequencing of 5S ribosomal ribonucleic acid suggests some degree of relatedness with all organisms now classified in the Mollicutes; (vi) strain BC-3T is capable of viscotactic and chemotactic responses; (vii) strain BC-3T possesses fibrils that may mediate various types of motility; and (viii) a lytic virus (SpV4) isolated from Spiroplasma sp. strain B63 (a representative of subgroup I-2) is morphologically and genomically distinct from other spiroplasma viruses and forms plaques only on lawns of subgroup I-2 spiroplasmas. Previous work on strain AS 576, another member of subgroup I-2, demonstrated (i) a viscotactic response, (ii) moderate sensitivity to osmotic environments, (iii) susceptibility to tetracycline and aminoglycoside antibiotics, (iv) growth in a relatively simple, chemically defined medium, (v) nutritional utilization patterns in defined medium, and (vi) a genome molecular weight of 109. On the basis of our new findings and the previously described properties of strain BC-3T and related subgroup I-2 strains, we propose that spiroplasma strains with the characteristics described here be classified as a new species, Spiroplasma melliferum. Strain BC-3, the type strain, has been deposited in the American Type Culture Collection as strain ATCC 33219.

Cultivation of the Drosophila sex-ratio spiroplasma.

Uncultivable for more than 25 years, the sex-ratio spiroplasma of Drosophila willistoni grew in a tissue culture medium (H-2) containing an embryo-derived lepidopteran cell line (IPLB-TN-R2). After adaptation, it grew in a cell-free H-2 medium. This success demonstrates the usefulness of cell culture systems for cultivation of fastidious microorganisms and facilitates study of the sex-ratio trait in Drosophila.

Revised group classification of the genus Spiroplasma

Significant changes have been made in the systematics of the genus Spiroplasma (class Mollicutes) since it was expanded by revision in 1987 to include 23 groups and eight sub-groups. Since that time, two additional spiroplasmas have been assigned group numbers and species names. More recently, specific epithets have been assigned to nine previously designated groups and three sub-groups. Also, taxonomic descriptions and species names have been published for six previously ungrouped spiroplasmas. These six new organisms are: Spiroplasma alleghenense (strain PLHS-1T) (group XXVI), Spiroplasma lineolae (strain TALS-2T) (group XXVII), Spiroplasma platyhelix (strain PALS-1T) (group XXVIII), Spiroplasma montanense (strain HYOS-1T) (group XXXI), Spiroplasma helicoides (strain TABS-2T) (group XXXII) and Spiroplasma tabanidicola (strain TAUS-1T) (group XXXIII). Also, group XVII, which became vacant when strain DF-1T (Spiroplasma chrysopicola) was transferred to group VIII, has been filled with strain Tab 4c. The discovery of these strains reflects continuing primary search in insect reservoirs, particularly horse flies and deer files (Diptera: Tabanidae). In the current revision, new group designations for 10 spiroplasma strains, including six recently named organisms, are proposed. Three unnamed but newly grouped spiroplasmas are strain TIUS-1 (group XXIX; ATCC 51751) from a typhiid wasp (Hymenoptera: Tiphiidae), strain BIUS-1 (group XXX; ATCC 51750) from floral surfaces of the tickseed sunflower (Bidens sp.) and strain BARC 1901 (group XXXIV; ATCC 700283). Strain BARC 2649 (ATCC 700284) from Tabanus lineola has been proposed as a new sub-group of group VIII. Strains TIUS-1 and BIUS-1 have unusual morphologies, appearing as helices at only certain stages in culture. In this revision, potentially important intergroup serological relationships observed between strain DW-1 (group II) from a neotropical Drosophila species and certain sub-group representatives of group I spiroplasmas are also reported.

Spiroplasma kunkelii sp. nov.: Characterization of the Etiological Agent of Corn Stunt Disease

Nine strains of spiroplasma subgroup 1-3, which comprise the etiological agent of corn stunt disease, were similar in their serological properties. Strain E275T(T = type strain) was studied by using criteria proposed by the International Committee on Systematic Bacteriology Subcommittee on Taxonomy of Mollicutes for descriptions of new mollicute species. This strain was shown to belong to the class Mollicutes by the ultrastructure of its limiting membrane, its procaryotic organization, its colonial morphology, and its filtration behavior and to the family Spiroplasmataceae by its helical morphology and motility. Although some serological cross-reactions with other group I spiroplasma strains was observed, strain E275Tcould be readily distinguished from representatives of other group I subgroups. Subgroup 1-3 spiroplasmas and other group I strains also differed in their one- and two-dimensional polyacrylamide gel protein patterns, plant and insect host ranges, and pathogenicities. Growth in MIA or MID medium occurred at 20 to 30°C. Cholesterol was required for growth. Glucose was fermented, and arginine was catabolized. Subgroup 1-3 strains, including strain E275T, reacted with considerable homogeneity in deformation tests and were completely separable from strains belonging to subgroup I-1 (Spiroplasma citri) and subgroup I-2 (Spiroplasma melliferum). Strain E275Twas also serologically distinct from subgroups I-4 through I-8, Spiroplasma floricola (group III), Spiroplasma apis (group IV), Spiroplasma mirum (group V), and representative strains of spiroplasma groups II and VI through XI. The deoxyribonucleic acid of strain E275Thybridized with the deoxyribonucleic acid of S. citri at significant levels (33 to 68%, depending on the technique used). These results demonstrate that strain E275Tand similar strains meet the criteria proposed by the International Committee on Systematic Bacteriology Subcommittee for elevation of spiroplasma subgroups to species. We propose that such strains be named Spiroplasma kunkelii. Strain E275Thas been deposited in the American Type Culture Collection as strain ATCC 29320T.

Revised group classification of the genus Spiroplasma (class Mollicutes), with proposed new groups XII to XXIII

Fourteen spiroplasma strains, primarily of insect origin, were analyzed according to criteria previously proposed for description of new serogroups of the genus Spiroplasma. When tested by reciprocal metabolism inhibition, growth inhibition, and deformation serological procedures, 12 of the strains were serologically unrelated to each other and to representative strains previously assigned to groups I to XI and subgroups I-1 to I-8. Examination by dark-field and electron microscopy indicated that each of the 12 strains possessed morphological features typical of spiroplasmas (helicity, motility, lack of a cell wall, and absence of periplasmic fibrils). All strains were resistant to 500 U of penicillin per ml and catabolized glucose but were unable to hydrolyze urea. Ability to hydrolyze arginine varied among strains. The guanine-plus-cytosine contents of the deoxyribonucleic acid of the 12 strains varied from 24 to 29 mol%. Two other strains (MQ-6 and Ar-1357) shared only a partial serological relationship to strain CC-1 (group XVI), suggesting that this group may consist of an assemblage of heterogeneous serovars. On the basis of the unique serological distinctions and other properties reported herein, we propose that the 12 representative strains be assigned new consecutive group designations XII to XXIII.

Spiroplasmas and Acholeplasmas: Multiplication in Insects

The helical wall-free microorganism, Spiroplasma citri, which is associated with citrus stubborn, a disease with no known vector, multiplied in the leafhopper vector of corn stunt but multiplied to higher titer in the vector of aster yellows and decreased the longevity of that insect. Acholeplasma laidlawii and A. granularum also multiplied in both leafhoppers.

Spiroplasma mirum, a New Species from the Rabbit Tick (Haemaphysalis leporispalustris)

Three spiroplasma strains recovered from rabbit ticks (Haemaphysalis leporispalustris) in Georgia and Maryland were found to be similar in biochemical, serological, and pathological properties. The organisms grew at temperatures of 20 to 37°C, required cholesterol for growth, fermented glucose, hydrolyzed arginine, and produced a film and spot reaction. The three spiroplasma strains were serologically distinct from the one established species (Spiroplasma citri) in the genus and from all other unclassified spiroplasma serogroups presently known. On the basis of these findings and other morphological, biological, and serological properties of the organism, it is proposed that spiroplasma strains with these characteristics be classified as a new species, Spiroplasma mirum. Strain SMCA (ATCC 29335) is the type strain.